A bird's-eye view of the endoplasmic reticulum in filamentous fungi.

SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Nanosized extracellular vesicles released by Neurospora crassa hyphae.

Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa culture's supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (dh) of 68 nm and a particle diameter (dp) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.

Multicolor light-sheet microscopy for a large field of view imaging: A comparative study between Bessel and Gaussian light-sheets configurations.

Light-sheet fluorescence microscopy (LSFM) is useful for developmental biology studies, which require a simultaneous visualization of dynamic microstructures over large fields of views (FOVs). A comparative study between multicolor Bessel and Gaussian-based LSFM systems is presented. Discussing the chromatic implications to achieve colocalized and large FOVs when both optical arrays are implemented under the same excitation objective is the purpose of this work. The light-sheets FOVs, optical sectioning, and resolution are experimentally characterized and discussed. The advantages of using Bessel beams and the main drawbacks of using Gaussian beams for multicolor imaging are highlighted. Multiple Bessel excitation minimizes the FOV's mismatch's effects due to the beams chromatic defocusing and alleviates the aside object blurring obtained with multiple Gaussian beams. It also offers a fair homogeneous axial resolution and optical sectioning over a larger effective FOV. Imaging over perithecia samples of the fungus Sordaria macrospora demonstrates such advantages. This work complements previous comparative studies that discuss only single wavelengths light-sheets excitations.

Chemical Diversity and Antimicrobial Potential of Cultivable Fungi from Deep-Sea Sediments of the Gulf of Mexico.

A collection of 29 cultivable fungal strains isolated from deep-sea sediments of the Gulf of Mexico were cultivated under the "one strain, many compounds" approach to explore their chemical diversity and antimicrobial potential. From the 87 extracts tested, over 50% showed antimicrobial activity, and the most active ones were those from cultures grown at 4 °C in darkness for 60 days (resembling deep-sea temperature). PCA analysis of the LC-MS data of all the extracts confirmed that culture temperature is the primary factor in the variation of the 4462 metabolite features, accounting for 21.3% of the variation. The bioactivity-guided and conventional chemical studies of selected fungal strains allowed the identification of several active and specialized metabolites. Finally, metabolomics analysis by GNPS molecular networking and manual dereplication revealed the biosynthetic potential of these species to produce interesting chemistry. This work uncovers the chemical and biological study of marine-derived fungal strains from deep-sea sediments of the Gulf of Mexico.

Trade-off between Plasticity and Velocity in Mycelial Growth.

Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity.IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

The metabolic core of the prokaryotic community from deep-sea sediments of the southern Gulf of Mexico shows different functional signatures between the continental slope and abyssal plain.

Marine sediments harbor an outstanding level of microbial diversity supporting diverse metabolic activities. Sediments in the Gulf of Mexico (GoM) are subjected to anthropic stressors including oil pollution with potential effects on microbial community structure and function that impact biogeochemical cycling. We used metagenomic analyses to provide significant insight into the potential metabolic capacity of the microbial community in Southern GoM deep sediments. We identified genes for hydrocarbon, nitrogen and sulfur metabolism mostly affiliated with Alpha and Betaproteobacteria, Acidobacteria, Chloroflexi and Firmicutes, in relation to the use of alternative carbon and energy sources to thrive under limiting growth conditions, and metabolic strategies to cope with environmental stressors. In addition, results show amino acids metabolism could be associated with sulfur metabolism carried out by Acidobacteria, Chloroflexi and Firmicutes, and may play a crucial role as a central carbon source to favor bacterial growth. We identified the tricarboxylic acid cycle (TCA) and aspartate, glutamate, glyoxylate and leucine degradation pathways, as part of the core carbon metabolism across samples. Further, microbial communities from the continental slope and abyssal plain show differential metabolic capacities to cope with environmental stressors such as oxidative stress and carbon limiting growth conditions, respectively. This research combined taxonomic and functional information of the microbial community from Southern GoM sediments to provide fundamental knowledge that links the prokaryotic structure to its potential function and which can be used as a baseline for future studies to model microbial community responses to environmental perturbations, as well as to develop more accurate mitigation and conservation strategies.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Of Mice and Fungi: Coccidioides spp. Distribution Models.

The continuous increase of Coccidioidomycosis cases requires reliable detection methods of the causal agent, Coccidioides spp., in its natural environment. This has proven challenging because of our limited knowledge on the distribution of this soil-dwelling fungus. Knowing the pathogen's geographic distribution and its relationship with the environment is crucial to identify potential areas of risk and to prevent disease outbreaks. The maximum entropy (Maxent) algorithm, Geographic Information System (GIS) and bioclimatic variables were combined to obtain current and future potential distribution models (DMs) of Coccidioides and its putative rodent reservoirs for Arizona, California and Baja California. We revealed that Coccidioides DMs constructed with presence records from one state are not well suited to predict distribution in another state, supporting the existence of distinct phylogeographic populations of Coccidioides. A great correlation between Coccidioides DMs and United States counties with high Coccidioidomycosis incidence was found. Remarkably, under future scenarios of climate change and high concentration of greenhouse gases, the probability of habitat suitability for Coccidioides increased. Overlap analysis between the DMs of rodents and Coccidioides, identified Neotoma lepida as one of the predominant co-occurring species in all three states. Considering rodents DMs would allow to implement better surveillance programs to monitor disease spread.

The Mycobiota of the Deep Sea: What Omics Can Offer.

The deep sea (>1000 m below sea level) represents one of the most extreme environments of the ocean. Despite exhibiting harsh abiotic conditions such as low temperatures, high hydrostatic pressure, high salinity concentrations, a low input of organic matter, and absence of light, the deep sea encompasses a great fungal diversity. For decades, most knowledge on the fungal diversity of the deep sea was obtained through culture-dependent techniques. More recently, with the latest advances of high-throughput next generation sequencing platforms, there has been a rapid increment in the number of studies using culture-independent techniques. This review brings into the spotlight the progress of the techniques used to assess the diversity and ecological role of the deep-sea mycobiota and provides an overview on how the omics technologies have contributed to gaining knowledge about fungi and their activity in poorly explored marine environments. Finally, current challenges and suggested coordinated efforts to overcome them are discussed.

Cultivable fungi from deep-sea oil reserves in the Gulf of Mexico: Genetic signatures in response to hydrocarbons.

The estimation of oil spill effects on marine ecosystems is limited to the extent of our knowledge on the autochthonous biota. Fungi are involved in key ecological marine processes, representing a major component of post-spill communities. However, information on their functional capacities remains lacking. Herein we analyzed cultivable fungi from sediments in two oil-drilling regions of the Gulf of Mexico for their ability to tolerate and use hexadecane and 1-hexadecene as the sole carbon sources; and to evaluate gene expression profiles of key hydrocarbonoclastic taxa during utilization of these hydrocarbons. The isolated fungi showed differential sensitivity patterns towards the tested hydrocarbons under three different concentrations. Remarkably, six OTUs (Aureobasidium sp., Penicillium brevicompactum, Penicillium sp., Phialocephala sp., Cladosporium sp. 1 and 2) metabolized the tested alkane and alkene as the sole carbon sources, confirming that deep-sea fungal taxa are valuable genetic resources with potential use in bioremediation. RNA-seq results revealed distinctive gene expression profiles in the hydrocarbonoclastic fungus Penicillium sp. when using hexadecane and 1-hexadecene as the sole carbon sources, with up-regulation of genes involved in transmembrane transport, metabolism of six-carbons carbohydrates, and nitric oxide pathways.

Comparative genomics reveals the origin of fungal hyphae and multicellularity.

Hyphae represent a hallmark structure of multicellular fungi. The evolutionary origins of hyphae and of the underlying genes are, however, hardly known. By systematically analyzing 72 complete genomes, we here show that hyphae evolved early in fungal evolution probably via diverse genetic changes, including co-option and exaptation of ancient eukaryotic (e.g. phagocytosis-related) genes, the origin of new gene families, gene duplications and alterations of gene structure, among others. Contrary to most multicellular lineages, the origin of filamentous fungi did not correlate with expansions of kinases, receptors or adhesive proteins. Co-option was probably the dominant mechanism for recruiting genes for hypha morphogenesis, while gene duplication was apparently less prevalent, except in transcriptional regulators and cell wall - related genes. We identified 414 novel gene families that show correlated evolution with hyphae and that may have contributed to its evolution. Our results suggest that hyphae represent a unique multicellular organization that evolved by limited fungal-specific innovations and gene duplication but pervasive co-option and modification of ancient eukaryotic functions.

Targeted ITS1 sequencing unravels the mycodiversity of deep-sea sediments from the Gulf of Mexico.

Fungi from marine environments have been significantly less studied than terrestrial fungi. This study describes distribution patterns and associated habitat characteristics of the mycobiota of deep-sea sediments collected from the Mexican exclusive economic zone (EEZ) of the Gulf of Mexico (GoM), ranging between 1000 and > 3500 m depth. Internal Transcribed Spacer 1 (ITS1) amplicons were sequenced by Illumina MiSeq. From 29 stations sampled across three annual campaigns, a total of 4421 operational taxonomic units (OTUs) were obtained, indicating a high fungal richness. Most OTUs assignments corresponded to Ascomycota, unidentified fungi and Basidiomycota. The majority of the stations shared a mere 31 OTUs, including the worldwide reported genera Penicillium, Rhodotorula and Cladosporium. Both a transient and a conserved community were identified, suggesting their dependence on or adaptation to the habitat dynamics, respectively. The differences found in fungal richness and taxonomic compositions were correlated principally with latitude, carbon and carbonates content, and terrigenous content, which could be the potential drivers that delimit fungal distribution. This study represents an expansion of our current knowledge on the biogeography of the fungal community from deep-sea sediments, and identifies the geographic and physicochemical properties that delimit fungal composition and distribution in the GoM.

Multicolor fluorescence microscopy using static light sheets and a single-channel detection.

We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.

Coccidioides ecology and genomics.

Although the natural history and ecology of Coccidioides spp. have been studied for over 100 years, many fundamental questions about this fungus remain unanswered. Two of the most challenging aspects of the study of Coccidioides have been the undefined ecological niche and the outdated geographic distribution maps dating from midcentury. This review details the history of Coccidioides ecological research, and discusses current strategies and advances in understanding Coccidioides genetics and ecology.

Off the wall: The rhyme and reason of Neurospora crassa hyphal morphogenesis.

The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, ß-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkörper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.